Journal of Bacteriology, March 2004, p . 1337-1344, Vol . 186, No . 5

The Pseudomonas putida Crc Global Regulator Controls the Expression of Genes from Several Chromosomal Catabolic Pathways for Aromatic Compounds

Gracia Morales,¹ Juan Francisco Linares,¹ Ana Beloso,² Juan Pablo Albar,² José Luis Martínez,¹ and Fernando Rojo^1*

Departamento de Biotecnología Microbiana,¹ Servicio de Proteómica, Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain²

Received 29 September 2003/ Accepted 26 November 2003

 
The Crc protein is involved in the repression of several catabolic pathways for the assimilation of some sugars, nitrogenated compounds, and hydrocarbons in Pseudomonas putida and Pseudomonas aeruginosa when other preferred carbon sources are present in the culture medium [catabolic repression] . Crc appears to be a componentof a signal transduction pathway modulating carbon metabolismin pseudomonads, although its mode of action is unknown . Tobetter understand the role of Crc, the proteome profile of twootherwise isogenic P . putida strains containing either a wild-typeor an inactivated crc allele was compared . The results showedthat Crc is involved in the catabolic repression of the hpdand hmgA genes from the homogentisate pathway, one of the central catabolic pathways for aromatic compounds that is used to assimilate intermediates derived from the oxidation of phenylalanine, tyrosine, and several aromatic hydrocarbons . This led us to analyze whetherCrc also regulates the expression of the other central catabolicpathways for aromatic compounds present in P . putida . It wasfound that genes required to assimilate benzoate through thecatechol pathway [benA and catBCA] and 4-OH-benzoate throughthe protocatechuate pathway [pobA and pcaHG] are also negativelymodulated by Crc . However, the pathway for phenylacetate appearedto be unaffected by Crc . These results expand the influenceof Crc to pathways used to assimilate several aromatic compounds,which highlights its importance as a master regulator of carbonmetabolism in P . putida.

 
Expression of bacterial catabolic pathways is usually tightly controlled . Regulation can respond to the presence or absenceof the compound to be assimilated [a specific control response]or to signals that link the induction of the pathway genes tothe physiological status of the cell [a global control response].The global control is normally dominant over the specific control.One such global control mechanism is the so-called catabolicrepression, a complex regulatory response that allows the cellto preferentially use a particular carbon source over a mixtureof several other potentially assimilable, but less preferred,compounds . Catabolic repression seems to operate through differentmechanisms in different bacterial species . In pseudomonads,this process has been studied for some pathways responsiblefor the assimilation of sugars, amino acids, hydrocarbons andaromatic compounds [for reviews, see references 4, 5, and 27]. However, the molecular mechanisms underlying catabolic repression in pseudomonads remain mostly unknown . The metabolism of hydrocarbons and aromatic compounds has attracted special attention because many of them are responsible for important pollution problemsin the environment . Available evidence indicates that expressionof the pathways for the assimilation of hydrocarbons and aromaticcompounds is modulated by different kinds of global controlsignals, with catabolic repression being just one of them [8, 27].

Very few proteins have been shown to participate in catabolic repression in pseudomonads . The first to be described, Crc [for "catabolite repression control"], is involved in the catabolic repression generated by succinate or lactate on the expressionof a number of genes implicated in the metabolism of some sugarsand nitrogenated compounds . In Pseudomonas aeruginosa, genes regulated by Crc include those encoding glucose-6-phosphate dehydrogenase, glucokinase, 6-phospho-gluconate dehydratase, 2-keto-3-deoxy-6-phosphogluconatealdolase, amidase, and the branched-chain keto acid dehydrogenase [5, 11, 20, 34] . At least some of these genes [glucose-6-phosphatedehydrogenase, amidase, and branched-chain keto acid dehydrogenase]are controlled by Crc in Pseudomonas putida as well [11] . When cells grow in a rich medium such as 2x YT or Luria-Bertani [LB],Crc also exerts a strong repression on the induction of theP . putida branched-chain keto acid dehydrogenase [11, 12] andof the alkane degradation pathway encoded in the OCT plasmidfrom P . putida GPo1 [36] . Available data suggest that Crc would be a component of a signal transduction pathway modulating carbon metabolism as well as other phenomena such as biofilm development [12, 21, 25] . Crc ultimately affects the expression of the regulatedgenes, although the precise molecular mechanism underlying thiseffect remains to be elucidated . Crc does not appear to bindDNA, suggesting that it is not a classical DNA-binding repressor,but its target has not been identified [5, 12, 20] . At leastin P . putida, the effect of Crc is particularly important incells growing exponentially in a rich medium [8, 12, 36].

In an effort to better understand the role of the Crc proteinon the regulation of bacterial metabolism, we have comparedthe proteome profile of a P . putida strain to that of an isogenic derivative in which the crc gene had been inactivated . This kind of global analysis had not been done before . The results indicate that Crc is involved in, among other things, the expression of genes belonging to several of the central chromosomal pathwaysfor the assimilation of aromatic compounds.

 
Bacterial strains and culture media. Strain PBS4 derives from P . putida KT2442 [9] by insertion inits chromosome of a PalkB::lacZ transcriptional fusion and of the alkS gene [35] . P . putida KT2442 is a rifampin-resistantderivative of P . putida KT2440 [9]. P . putida PBS4C1 derivesfrom strain PBS4 by inactivation of the crc gene [contains acrc::tet allele] [36] . Strains were grown at 30°C in LBmedium [29] supplemented where indicated with 5 mM 4-hydroxybenzoate,5 mM benzoate, or 5 mM phenylacetate . Cell growth was monitoredby measuring turbidity at 600 nm.

Two-dimensional [2-D] electrophoresis and mass spectrum analysis. Twenty milliliters of exponentially growing cells [A₆₀₀ of 0.6]were spun down at 4°C; resuspended in 60 µl of 0.3% [wt/vol] sodium dodecyl sulfate [SDS], 5% [vol/vol] ß-mercaptoethanol, and 50 mM Tris-HCl [pH 8]; and boiled for 2 min . Samples were treated for 30 min on ice with a DNase I-RNase solution [final concentrations, 15 mg of DNase I/ml, 75 mg of RNase A/ml, 1mM MgCl₂] . Finally, 240 µl of a lysis buffer containing6 M urea, 2 M thiourea, 4% [vol/vol] 3-[[3-cholamidopropyl]-dimethylammonio]-1-propanesulfonate [CHAPS], 1% [vol/vol] precast pH gradient buffer [pH 4 to 7; Amersham Biosciences, Uppsala, Sweden] and 2 mM Tris carboxyethyl phosphine-HCl was added.

2-D electrophoresis was performed as described previously [10, 26] with precast immobilized pH 4 to 7 gradient [IPG] strips[18 cm in length; Amersham Biosciences] for the first dimension[isoelectric focusing [IEF]] . Briefly, 100-µg protein samples were applied by anodic cup-loading of IPG strips previously rehydrated with 350 µl of rehydration buffer [6 M urea,2 M thiourea, 2% [wt/vol] CHAPS, 0.5% [vol/vol] of the corresponding carrier ampholytes, 1 mM Tris carboxy ethyl phosphine-HCl, andminute amounts of bromophenol blue] for at least 10 h . Focusingwas carried out with the IPG Phor [Amersham Biosciences] byapplying an increasing voltage as follows: 200 V for 1 h; gradientincreases from 200 to 3,000 V for 3 h, 3,000 to 6,000 V for2 h, and 6,000 to 8,000 V for 1 h; and constant 8,000 V untila total of 60,000 V · h was reached . After IEF separation,the strips were equilibrated two times for 20 min with 50 mMTris-HCl [pH 8.8], 6 M urea, 30% [vol/vol] glycerol, 2% [wt/vol]SDS, and trace amounts of bromophenol blue . The first equilibrationsolution contained 2% [wt/vol] dithiothreitol . The second equilibrationsolution contained 4% [wt/vol] iodoacetamide . The second-dimension[SDS-polyacrylamide gel electrophoresis] was performed with1-mm-thick, 16- by 15-cm, 12.5, 10, or 8% [wt/vol] homogeneouspolyacrylamide gels, and electrophoresis was carried out overnightat constant current [5 mA/gel] and temperature [5°C] . Afterelectrophoresis, gels were stained with the mass spectrometry-compatiblemodified PlusOne silver-staining protein kit [Amersham Biosciences].

In-gel digestion of proteins and matrix-assisted laser desorption ionization [MALDI] peptide mass fingerprinting. Protein spots were excised manually and then processed automaticallywith an Investigator ProGest protein digestion station [GenomicSolutions, Huntingdon, Cambridgeshire, United Kingdom] [16]. The digestion protocol used was as described earlier [30] withminor variations . Gel plugs were washed with 25 mM ammonium bicarbonate and acetonitrile prior to reduction with 10 mM dithiothreitol in 25 mM ammonium bicarbonate, and alkylation was done with100 mM iodoacetamide in 50 mM ammonium bicarbonate . The gelpieces were then rinsed with 50 mM ammonium bicarbonate andacetonitrile and dried under a stream of nitrogen . Modifiedporcine trypsin [sequencing grade; Promega, Madison, Wis.] ata final concentration of 16 ng/µl in 25 mM ammonium bicarbonatewas added to the dry gel pieces, and the digestion proceededat 37°C for 12 h . Peptides were eluted with 25 mM ammoniumbicarbonate and 0.1% [vol/vol] trifluoroacetic acid for a finalextraction volume of 50 µl.

For MALDI peptide mass fingerprinting, a 0.3-µl aliquotof matrix solution [5 g of 2,5-dihydroxybenzoic acid/liter in33% [vol/vol] aqueous acetonitrile and 0.1% [vol/vol] trifluoroaceticacid] was manually deposited onto a 400-µm-diameter AnchorChipMALDI target and allowed to dry at room temperature . Then, 0.3µl of the above-described extraction solution was addedand allowed to dry at room temperature . Samples were measuredon a Reflex IV MALDI-time of flight mass spectrometer [Bruker-FranzenAnalytic GmbH, Bremen, Germany] equipped with the SCOUT sourcein positive-ion reflector mode with delayed extraction . Theion acceleration voltage was 20 kV . The equipment was firstexternally calibrated by employing protonated mass signals froma peptide mixture covering the 1,000 to 3,500 m/z range, andthereafter, every spectrum was internally calibrated by usingselected signals arising from trypsin autoproteolysis to reacha typical mass measurement accuracy of ±30 ppm . The measuredtryptic peptide masses were transferred through the BioToolsprogram as inputs to search either the National Center for BiotechnologyInformation nonredundant database or a P . putida KT2440 database[23] by using Mascot software [Matrix Science, London, UnitedKingdom] . No restrictions were placed on the species of originof the protein, and the allowed protein molecular mass was 1to 200 kDa . Up to one missed tryptic cleavage was considered,and a mass accuracy of 50 ppm was used for all tryptic masssearches.

RT-PCR assays. Exponentially growing cells [A₆₀₀ of 0.6] were collected, spundown at 4°C, and frozen in dry ice at -80°C . Total RNAwas extracted by using the phenol-guanidine thiocyanate mixTri Reagent LS [Molecular Research Center, Inc.] . Residual DNAwas removed by treatment with DNase I . Reverse transcriptase[RT]-PCR assays were performed by using Ready-To-Go RT-PCR beads[Amersham Biosciences] as indicated by the manufacturer, primersspecific for the desired genes, and serial 10-fold dilutions of the RNA [1, 0.1, and 0.01 µg] to ensure a linear response. To ascertain that no residual DNA was present in the RNA preparations, a PCR was performed with the same primers and overall conditions, except that no RT was added.

Determination of benzoate, 4-OH-benzoate, and phenylacetate. Benzoate, 4-OH-benzoate, and phenylacetate utilization by wholecells was monitored by measuring their concentrations in culture supernatants by high-performance liquid chromatography [HPLC].The column used was a reverse-phase octyldecyl silane hypersilC₁₈ [124 by 4 mm], and the mobile phase contained 60% [vol/vol]11 mM H₃PO₄ and 40% [vol/vol] methanol . The absorbance of theeluate was monitored at 254 nm.

 
Influence of Crc on the proteome of P . putida cells growing exponentially in LB medium. To have a global view of the effect of the Crc regulator onthe expression of the P . putida proteome, the total proteinsof P . putida strain PBS4 and of its isogenic crc mutant derivativePBS4C1 were analyzed by 2-D electrophoresis . Cells were collectedwhile actively growing in rich medium [A₆₀₀ = 0.6], since, atleast in the case of the OCT plasmid alkane degradation pathway,the repression effect of Crc is observed only during the exponentialphase of growth [36] . Inactivation of the crc gene led to aclear increase in the intensity of 11 protein spots in the 2-Dgels and to a decrease in the amount of two proteins [of 315spots detected] . Figure 1 shows selected areas of the 2-D gelswhere such changes were detected; only those spots whose intensityreproducibly changed in at least three independent assays aremarked . To identify the nature of the spots whose intensitiesvaried by inactivation of crc, each spot was excised from thestained gels and digested with trypsin, and the peptides generatedwere resolved by MALDI-time of flight mass spectrometry . Thepeptide patterns were compared to those of a virtual digestionof each protein encoded by the P . putida KT2440 genome, whosesequence has been determined [23] [www.tigr.org], or to digestionsof the proteins included in the National Center for Biotechnology Information nonredundant database by using the MASCOT software.

 
As detailed in Table 1, spots 1 and 2, which where present in the crc⁺ strain but absent in the crc mutant strain, showedthe highest scores with P . putida KT2440 cysteinyl-tRNA synthetase[CysS] and malate-quinone oxidoreductase 2 [Mqo-2], respectively.The P . putida KT2440 genome encodes three malate-quinone oxidoreductases[Mqo-1, Mqo-2, and Mqo-3], which are very similar in sequence[23] . Malate-quinone oxidoreductase is an enzyme of the citricacid cycle-glyoxylate cycle, and it transforms malate into oxaloacetate.In Escherichia coli, the activity of malate-quinone oxidoreductaseis regulated by the global regulator ArcA, the carbon sourceused, and by the growth phase [33] . Its regulation in pseudomonads is not so well characterized . In the case of P . aeruginosa, this enzyme has been shown to be essential for growth at theexpense of ethanol or acetate [18] . Our results show that the levels of Mqo-2 in P . putida are under the influence of Crc.

 
Spots 3 and 4, which were visible only when the crc gene was inactivated, were both identified as homogentisate 1,2 dioxygenase [HmgA] . Therefore, the two spots are probably isoforms of thesame enzyme with a slightly different pI . HmgA is the key enzymeof the homogentisate pathway, one of the central pathways forthe catabolism of aromatic compounds in P . putida and severalother bacteria [17] [Fig . 2] . Interestingly, spots 11 and 12,which were also visible only in the gel corresponding to the crc mutant strain, were identified as two isoforms of 4-hydroxyphenylpyruvatedioxygenase [Hpd] . This enzyme hydroxylates 4-hydroxyphenylpyruvateto render homogentisate, which is then cleaved by HmgA [Fig.2] . The hpd and hmgA genes map separately in the P . putida KT2440 chromosome [17] . It is worth noting that the aromatic amino acids phenylalanine and tyrosine, which are potential carbon and nitrogen sources considering that cells were grown in LBmedium, are metabolized through the homogentisate pathway afterconversion to 4-hydroxyphenylpyruvate [Fig . 2] [17].

 
Spot 5, whose intensity increased about threefold upon inactivation of crc, showed the highest homology to BraC . In P . aeruginosa, this protein has been characterized as the periplasmic amino acid-binding component of the high affinity LIV-I transportsystem for alanine, threonine, and branched-chain amino acids[13] . This transport system is encoded by the braCDEFG operon[14] . In both P . putida and P . aeruginosa, growth in the presence of branched-chain amino acids induces the expression of the bkd operon, which encodes a keto acid dehydrogenase that allows their assimilation [32] . Expression of this operon is regulatedby catabolite repression, an effect that is at least in partmediated by the Crc protein [11] . Induction of the bkd operonin a crc mutant strain grown in a rich medium is, however, low,even if the medium is supplemented with valine and isoleucine[12] . This probably explains why we were unable to detect thepolypeptides of the branched-chain keto acid dehydrogenase inour 2-D gels . However, our results clearly show that Crc controlsthe expression of the transport system for branched-chain aminoacids, an observation that, to our knowledge, had not been reportedbefore.

Spots 6 and 9 corresponded to two isoforms of open reading frame PP1015, identified as the periplasmic sugar-binding componentof a sugar ABC transporter . Spot 6 increased by about eightfoldupon inactivation of crc, whereas spot 9 was almost undetectablein the strain containing a wild-type crc allele . As detailedin the introduction, Crc is involved in the repression of anumber of genes implicated in the oxidation of some sugars inP . aeruginosa and P . putida . Our finding that Crc also regulatesthe expression of components of the sugar transporters is consistentwith these observations and highlights the importance of Crcin the regulation of carbohydrate metabolism in P . putida . The precise role of regulation of open reading frame PP1015 has,to our knowledge, not been reported.

Spots 7 and 8 were identified as two isoforms of OadA, the alpha subunit of oxaloacetate decarboxylase . This enzyme catalyzesthe decarboxylation of oxaloacetate to pyruvate and CO₂ [7]. Oxaloacetate decarboxylase is formed by three subunits, alpha, beta, and gamma, encoded by the oadGAB genes [19] . This enzymehas been studied mainly in Klebsiella pneumoniae, where it participatesin citrate fermentation under anaerobic conditions . Expressionof the oxaloacetate decarboxylase in K . pneumoniae is subjectto catabolite repression by the CRP protein [22] . A proteinshowing high similarity to E . coli and K . pneumoniae CRP ispresent in P . aeruginosa and in P . putida and has been calledVfr [1] . Evidence gathered to date indicates that Vfr is a globalregulator of gene expression . However, it is not involved incatabolite repression but in regulation of the quorum-sensingresponse [1, 31] . It is interesting that expression of oxaloacetate decarboxylase is regulated by catabolic repression in both P. putida and K . pneumoniae but through different global regulatoryproteins . This observation agrees with the idea that there areprobably diverse alternative strategies for reaching the same final regulatory response, with the only important requisite being that they all allow for a suitable responsiveness to theproper specific and global regulation signals [3].

Spots 10 and 13 corresponded to subunits A and B, respectively,of a probable coenzyme A [CoA] transferase, whose role is unknownat present.

Among the spots whose intensity varied upon inactivation ofcrc, we did not detect those of several proteins that are knownto be regulated by Crc, such as branched-chain keto acid dehydrogenase, glucose-5-phosphate dehydrogenase, and amidase [11, 12] . Thisresult is to be expected for proteins which are present in amountsbelow detection limits, which are not induced under the growthconditions used, or which have a pI or a molecular mass thatfalls outside the range resolved by the 2-D gels used.

Influence of Crc on expression of the P . putida aromatic catabolic pathways. The aerobic catabolism of aromatic compounds follows a numberof convergent pathways that lead to formation of a few key centralintermediates that are subsequently cleaved by specific dioxygenaseenzymes [Fig . 2] . In P . putida KT2440, the identified chromosomallyencoded aromatic pathways are the homogentisate pathway, thecatechol pathway, the protocatechuate pathway, and the phenylacetatepathway [17] . The catechol pathway eventually converges intothe protocatechuate pathway [Fig . 2] . The proteomic analysisdescribed above indicated that Crc represses the expressionof Hpd and HmgA from the homogentisate pathway . It is conceivablethat Crc could also affect other catabolic pathways for aromaticcompounds . Expression of these pathways is induced by the correspondingsubstrates [or their metabolites] . The cells utilized in theproteomic analyses described above were grown in LB medium,so that the amino acids phenylalanine and tyrosine that canbe obtained from it allow induction of the homogentisate pathway[Fig . 2] . However, the catechol, protocatechuate, and phenylacetatepathways are not expected to be active in this growth mediumunless the proper substrates are added, making it unlikely thatspots corresponding to these pathways can be visualized in the2-D gels shown in Fig. 1.

To analyze whether the catechol, protocatechuate, and phenylacetate catabolic pathways are also under the influence of Crc, strains PBS4 and PBS4C1 were grown in LB medium in the absence or presenceof either 5 mM benzoate [catabolized through the catechol pathway][Fig. 2], 5 mM 4-hydroxybenzoate [catabolized through the protocatechuatepathway], or 5 mM phenylacetate [catabolized through the phenylacetatepathway] . When cultures reached a turbidity of 0.6 [mid-exponentialphase], total RNA was obtained and the level of expression ofthe genes encoding key enzymes of the mentioned catabolic pathwayswas analyzed by RT-PCR . To compare the expression levels ofeach gene in the two strains used, the RT-PCR was performed with serial dilutions of the total RNA purified, and the RNA levels of a crc-independent gene were analyzed in parallel as an external control . The npt gene encoding resistance to kanamycin was used for this purpose, since it is present in both strains and is expressed at constant levels from a -10 extended promoter recognized by the vegetative RNA polymerase . As a first approach,the mRNA levels of the hpd and hmgA genes were analyzed in cells growing in LB medium . The proteomic analyses described above had indicated that the levels of the Hpd and HmgA proteins are undetectable in the strain containing a functional Crc proteinbut increase considerably upon inactivation of the crc gene. However, it was not known whether Crc should influence transcription of hpd and hmgA . The RT-PCR analysis showed that the mRNA levels corresponding to hpd and hmgA were clearly higher in the crcmutant strain than in the parental strain [Fig . 3] . This resultsuggests that Crc regulates the levels of Hpd and HmgA proteinsby interfering directly or indirectly with the transcriptionof the corresponding genes . The hmgA gene maps immediately upstreamfrom the fah and mai genes, which encode enzymes that transformthe product of homegentisate cleavage into acetoacetate andfumarate [Fig . 2 and 3] . RT-PCR assays showed that Crc controlsmai expression as well [Fig . 3] . To our knowledge, a detailed analysis of the expression of the hmgA, fah, and mai genes hasnot been reported . However, it would not be surprising to findthat they are cotranscribed . If this was the case, Crc controls expression of the three genes.

 
Growth of P . putida PRS2000 in a minimal salts medium containing benzoate as a carbon source leads to the induction of the benABC, benD, and catBCA genes [15] . BenABC and benD encode a benzoatedioxygenase and a dehydrogenase that converts benzoate intocatechol, which is further transformed by the products of thecatBCA genes to render ß-ketoadipate-enol-lactone[Fig . 2] . Expression of benABC is induced by benzoate by meansof the BenR transcriptional activator [6], and expression of catBCA is activated by the CatR activator in the presence of cis,cis-muconate, which is produced from catechol by the action of CatA . These genes are also present in the P . putida KT2440 genome [17] . The benA, catA, catB, and catC genes were selectedfor RT-PCR analysis . As shown in Fig . 3, the mRNA levels correspondingto these four genes were considerably higher in the crc mutantstrain PBS4C1 than in the parental strain PBS4, suggesting thatCrc regulates their expression, exerting an inhibitory effectwhen cells are grown in LB medium containing benzoate.

Assimilation of 4-hydroxybenzoate by P . putida PRS2000 requires the expression of pobA, which encodes a hydroxylase that transforms 4-hydroxybenzoate into protocatechuate; the enzymes encodedby the pca genes further transform protocatechuate into acetyl-CoA and succinyl-CoA [28] [Fig . 2] . The pobA gene is present inmany Pseudomonas and Acinetobacter strains, where its expressionis activated by the PobR [or PobC] activator in the presenceof 4-hydroxybenzoate [reference 2 and references therein] . Thepca genes are arranged in four different clusters, pcaHG, pcaBDC, pcaIJ, and pcaF [reference 28 and references therein] . Withthe exception of pcaHG, which is induced by protocatechuate,the remaining genes of the regulon are induced by ß-ketoadipatethrough the PcaR transcriptional regulator [28] . All these genesare present in P . putida KT2440 [17] . As shown in Fig . 3, themRNA levels corresponding to pobA, pcaH, and pcaG were clearlyhigher in the crc mutant strain PBS4C1 than in the parentalstrain PBS4 . Therefore, pobA and pcaHG apparently belong tothe Crc regulon as well.

Phenylacetate is produced from the oxidation of several other related compounds by a number of genes that conform to the phenylacetyl-CoA catabolon [24] . Assimilation of phenylacetate requires 14 genesorganized in three contiguous operons, the expression of whichis induced in the presence of phenylacetate [24] . The possibleinfluence of Crc on expression of the phenylacetate pathwaywas also investigated, monitoring the mRNA levels of the phaEand phaA genes, which specify the phenylacetyl-CoA ligase andthe enoyl-CoA hydratase isomerase I, respectively . Expressionof these two genes was rather similar in both the absence andpresence of Crc [Fig . 3], which suggests that Crc does not controltheir expression . However, the mRNA levels detected under theexperimental conditions used were very low . Therefore, conclusionson the expression of these genes should be made with caution[see below].

Influence of Crc on the assimilation of benzoate, 4-OH-benzoate, and phenylacetate. As a final way to investigate the influence of Crc on the catechol,protocatechuate, and phenylacetate pathways, the ability ofcells containing a wild-type or an inactivated crc allele toassimilate these compounds was determined . To this end, strainsPBS4 and PBS4C1 were grown in LB medium supplemented with benzoate[assimilated through the catechol pathway], 4-hydroxybenzoate[assimilated through the protocatechuate pathway], or phenylacetate.Exponentially growing cells were collected and resuspended inLB supplemented with the corresponding aromatic compound ata concentration of 5 mM . The consumption of each aromatic compoundwas monitored as a function of time by HPLC . As shown in Fig.4, the wild-type strain PBS4 was very inefficient at removingbenzoate from the culture medium, since 87% of the benzoatestill remained in the culture supernatant after a 90-min incubation.However, in the case of the crc mutant strain PBS4C1, only 30%of the initial benzoate could be detected after the same incubationtime . One hour later [minute 150], all benzoate had been consumedby strain PBS4C1, whereas in the case of the wild-type strain,about 75% of the compound remained unused in the culture supernatant.This result is consistent with the RT-PCR assays, which indicatedthat in LB medium, the presence of benzoate leads to efficientactivation of the benA and catA genes only in the crc mutantstrain and not in the parental strain.

 
Inactivation of Crc clearly also stimulated the removal of 4-OH-benzoate from the culture media, although its consumption was somewhat slower [Fig . 4] . This is again consistent with the RT-PCR assayresults shown in Fig . 3 . Under the same conditions, phenylacetatewas metabolized by neither the wild-type nor the crc mutantstrain [Fig . 4] . Both of them, however, could efficiently growin a minimal salts medium containing phenylacetate as the solecarbon source . These results suggest that the phenylacetatepathway is induced poorly, if at all, in cells growing in LBsupplemented with phenylacetate, an idea that agrees with thelow expression of the phaA and phaE genes observed in the RT-PCRassays whose results are shown in Fig. 3 . In summary, the metabolismof phenylacetate in LB medium seems to be inhibited, possiblyby catabolite repression, although Crc does not seem to be involvedin the process.

The work presented here shows that the expression of key genesto assimilate 4-hydroxyphenyl pyruvate, benzoate, and 4-OH-benzoate through the homogentisate, catechol, and protocatechuate pathways, respectively, is controlled by the Crc global regulatory proteinin P . putida . However, the pathway for phenylacetate does not seem to be regulated by Crc . The assimilation of many different aromatic compounds converges to the homogentisate, catechol,and protocatechuate pathways, both in P . putida KT2440 [17] and in many other Pseudomonas strains . These compounds add to the increasing list of hydrocarbons [36], sugars [5], and aminoacids [11, 12] that are not preferred carbon sources for P.putida and whose metabolism is inhibited when other preferredcarbon sources are available . Crc stands, therefore, as a masterregulator of carbon metabolism in P . putida in response to physiologicaland environmental inputs.

 
We are grateful to Eduardo Diaz for helpful discussions andfor help with Fig . 2 and to L . Yuste for excellent technical assistance.

This work was supported by grants BIO2000-0939 and GEN2001-4698-C05-01 from the Spanish Ministry of Science and Technology and grant CAM 07M/0120/2000 from Comunidad Autónoma de Madrid.J.F.L . was the recipient of a predoctoral fellowship from theSpanish Ministry of Science and Technology.

 
* Corresponding author . Mailing address: Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain . Phone: 34 91 585 45 39 . Fax: 34 91 585 45 06 . E-mail: frojo@cnb.uam.es .

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